Bypass graft surgery is limited by stenosis of vein grafts. Neointimal formation in vein graft stenosis is affected by oxidative stress, acute inflammatory response, and proliferation. Gene therapy offers a novel treatment strategy for vein graft stenosis because gene transfer can be done ex vivo during the graft operation. In this study we used adenovirus-mediated ex vivo gene transfer of extracellular superoxide dismutase (EC-SOD) alone or in combination with tissue inhibitor of metalloproteinase-1 (TIMP-1) or vaccinia virus antiinflammatory protein 35K to prevent vein graft stenosis in a jugular vein graft model in normocholesterolemic New Zealand White rabbits. Vein grafts were analyzed 14 and 28 days after the gene transfer, using histological methods. It was found that at the 2-week time point EC-SOD + 35K and EC-SOD + TIMP-1 combinations delivered by gene transfer were the most efficient treatments in decreasing neointimal formation. At the 4-week time point the effect was seen only in the EC-SOD + TIMP-1 combination group. The combination of antiinflammatory proteins (EC-SOD + 35K) was the most effective in reducing macrophage accumulation, which was still significant at the 4-week time point, but this did not prevent vein graft thickening. In conclusion, oxidative, inflammatory, and proliferative processes are important for neointimal formation in vein graft stenosis. In the rabbit model of vein graft disease, combination gene therapy with antioxidative, antiinflammatory, and antiproliferative genes was effective in decreasing neointimal formation. This may be because two different genes may more efficiently affect different pathogenetic pathways at the early stage of the disease process than gene transfer approaches based on single genes.